Identification, RNA splice defect assessment and AON correction of non-coding variants of ABCA4 in Stargardt disease

Abstract

Purpose : In ~35% of persons with autosomal recessive (ar) Stargardt disease (STGD1), no or one variant was identified in the protein coding regions of ABCA4. In 84 STGD1 cases with one or no ABCA4 variants, we searched for rare deep-intronic variants, assessed their effect on transcripts, and corrected selected splice defects by using antisense oligonucleotides (AONs).

Methods : Haloplex-based whole ABCA4 gene sequencing was performed in 84 STGD1 cases with one or no ABCA4 variant. In silico prediction of splice abnormalities was assessed by using 5 different algorithms, and the effect of the candidate disease variants was tested in vitro by transfecting minigenes splice constructs into HEK293T cells. Fibroblasts of STGD1 cases were reprogrammed into induced pluripotent stem cells and differentiated into photoreceptor precursor cells (PPCs). The effect of deep-intronic variants on RNA splicing was assessed by reverse transcription (RT)-PCR of PPC mRNA. AONs were designed and added to the medium of minigene-transfected HEK293T and PPCs and the effect was analyzed at the mRNA level.

Results : Analysis of sequence data yielded 355 rare variants in 84 probands. In silico predictions revealed putative splice abnormalities for 36 variants, and so far, 3 splice abnormalities were found for 11 tested variants. One variant activated a cryptic splice acceptor site and upon HEK293T transfection of the mutant minigene led to a pseudoexon (PE) insertion. It was found in 6 alleles of 5 unsolved Dutch STGD1 cases but not in 195 unsolved German STGD1 cases. Another variant generated a cryptic splice donor site and also led to a PE insertion. It was found in one additional Dutch STGD1 case, but not in the German STGD1 cohort. Another previously identified deep-intronic variant resulted in the insertion of a sizeable PE, which was confirmed in patient-derived PPCs. By treatment with AONs we corrected the splice defects of two of the three tested variants.

Conclusions : Whole ABCA4 gene sequencing and in vitro and in vivo assessments of the effect of rare deep-intronic variants enabled us to identify several novel STGD1-associated variants. AON delivery resulted in a correction of several splicing defects, showing that RNA modulation may represent an exciting therapeutic approach for Stargardt disease.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.